REGULATION OF CXC CHEMOKINE EXPRESSION BY H. PYLORI

描述（申请人提供）：幽门螺杆菌（Hp）感染是胃炎、消化性溃疡疾病和胃癌的主要原因。最近的临床和体外数据表明，Hp外炎症蛋白、OipA和cag致病性岛（PAI）通过诱导胃上皮细胞中白细胞介素(IL)-8的能力参与了疾病的发病。我们的长期目标是了解OipA单独或与cag PAI一起在hp相关疾病发病机制中的作用。在AIM 1中，我们将确定OipA单独或与cag PAI一起调节IL-8基因转录的体外分子机制。在hp感染的胃上皮细胞中，转录因子核因子-:B （NF-:B）、激活蛋白1 （AP-1）、干扰素刺激响应元件（ISRE）样元件和可能的干扰素-3激活序列（GAS）的结合位点参与调节IL-8基因转录。我们将使用紧密连接的极化胃上皮细胞（NCI-N87细胞）来克服非极化细胞的局限性。NCI-N87细胞使我们能够从顶膜（感染早期）和基底外膜（紧密连接开放或上皮屏障损伤后的晚期）检测不同的细胞信号通路。我们将重点关注细胞表面受体表皮生长因子受体（EGFR）和G蛋白偶联受体（GPCR）。Hp感染的体外模型将用于阐明1)导致IL-8基因转录的细胞表面受体的整体信号通路，2)OipA与cag PAI之间的相互作用，以及3)OipA负责IL-8基因转录的结构和基序。在AIM 2中，我们将利用蒙古沙鼠和gp130F759敲入小鼠模型，确定OipA和cag PAI在胃损伤中体外和体内作用的相关性。在沙鼠中，Hp感染诱导的胃粘膜转录因子在感染的阶段和作用方面是不同的（例如，AP-1是早期诱导的，导致炎症和溃疡，而NF- B和ISRE是晚期诱导的，导致萎缩）。我们之前的结果是基于对整个胃粘膜的分析。在这里，我们打算利用分离的胃上皮细胞来确定OipA和cag PAI如何诱导CXC趋化因子。我们还将使用IL-6家族共受体gp130上缺乏Src同源2磷酸酶（SHP-2）结合位点的Src同源2结构域的小鼠。在这些小鼠中，信号换能器和转录激活因子（STATs）信号过度激活，SHP-2->AP-1信号缺失。我们的初步数据显示Hp感染导致该小鼠模型的高增生性肿瘤，将进一步阐明OipA和cag PAI在胃十二指肠发病中的作用。我们假设，早期阶段，上皮细胞-细胞屏障在体外完好无损，将与体内急性炎症相关，而晚期阶段，上皮屏障在体外被破坏，将与慢性炎症、溃疡和致癌相关（即，当胃上皮结构在体内被破坏时）。本应用程序中描述的创新重点和战略方法将使人们对Hp感染生物学的理解达到新的水平。

DESCRIPTION (provided by applicant): Helicobacter pylori (Hp) infection is the major cause of gastric inflammation, peptic ulcer disease, and gastric cancer. Recent clinical and in vitro data indicate that the Hp outer inflammatory protein, OipA and the cag pathogenicity island (PAI) are involved in disease pathogenesis based on their ability to induce interleukin (IL)-8 in gastric epithelial cells. Our long term goal is to understand the role(s) of OipA, alone and together with the cag PAI, in the pathogenesis of Hp-related diseases. In AIM 1, we will determine in vitro molecular mechanisms by which OipA, alone and together with the cag PAI, regulate IL-8 gene transcription. Binding sites for the transcription factors nuclear factor-:B (NF-:B), activator protein 1 (AP-1), interferon-stimulated responsive element (ISRE)-like element, and likely interferon-3 activation sequence (GAS) in the IL-8 promoter are involved in regulating IL-8 gene transcription in Hp-infected gastric epithelial cells. We will use polarized gastric epithelial cells with tight junctions (NCI-N87 cells) to overcome the limitations of non-polarized cells. NCI-N87 cells allow us to detect different cell signaling pathways from the apical membrane (early phase of infection) and basolateral membranes (late phase following opening of tight junctions or epithelial-barrier damage). We will focus on the cell surface receptors epidermal growth factor receptor (EGFR) and G protein coupled receptor (GPCR). In vitro models of Hp infection will be used to clarify 1) overall signaling pathways from the cell surface receptor(s) resulting in IL-8 gene transcription, 2) interactions between OipA and the cag PAI, and 3) OipA structures and motifs responsible for IL-8 gene transcription. In AIM 2, we will determine the correlation between in vitro - in vivo roles of OipA and the cag PAI in gastric injury using Mongolian gerbils and the gp130F759 knock-in mouse model. In gerbils, gastric mucosal transcription factors induced by Hp infection differ with respect to phase and effect on infection (e.g., AP-1 is induced early and results in inflammation and ulceration whereas NF- B and ISRE are induced late and result in atrophy). Our previous results are based on analysis of the whole gastric mucosa. Here, we propose to determine how OipA and the cag PAI induce CXC chemokines using isolated gastric epithelial cells. We will also use mice that lack the Src homology 2 domain of the Src homology 2 phosphatase (SHP-2) binding site on the IL-6 family co- receptor, gp130. In these mice, the signal transducer and activator of transcriptions (STATs) signaling is hyperactivated and SHP-2->AP-1 signaling is absent. Our preliminary data showing that Hp infection results in hyperproliferative tumors in this mouse model will be extended to further clarify the roles of OipA and the cag PAI in the gastroduodenal pathogenesis. We hypothesize that the early phase, where the epithelial cell-cell barrier is intact in vitro, will correlate with acute inflammation in vivo and the late phase, where the epithelial barrier is disrupted in vitro, will correlate with chronic inflammation, ulceration and carcinogenesis (i.e., when the architecture of the gastric epithelium is disrupted in vivo). The innovative focus and strategic approaches described in this application will yield new levels of understanding of the biology of Hp infections. PUBLIC HEALTH RELEVANCE: Helicobacter pylori infection causes gastric inflammation, peptic ulcer disease, and gastric cancer. The work outlined in this application will use human gastric cells and animal models to identify the molecular mechanisms by which Helicobacter pylori virulence factors cause gastric inflammation and subsequent disease. This project will be able to identify novel therapeutic targets for the treatment of Helicobacter pylori infections and prevention of Helicobacter pylori-related diseases.